Saturation mutagenesis genome engineering of infective FX174 bacteriophage via unamplified oligo pools and golden gate assembly

Matthew S. Faber; James T. Van Leuven; Martina M. Ederer; Yesol Sapozhnikov; Zoë L. Wilson; Holly A. Wichman; Timothy A. Whitehead; Craig R. Miller

The gene fragments -F1, F2, G1, G2 -in the pCR2.1-TOPO plasmid (Supplemental Notes S1 and S2) had the BbvCI nicking site introduced via overhang PCR, type I restriction enzyme cutting, and ligation. First, PCR was performed with overhang primers (Supplemental Table S1 ) to introduce the BbvCI site. Standard Phusion Polymerase HF reaction conditions were used with 4 ng of template DNA, cycling conditions as follows: 98°C -1 min, 25x cycles of: 98°C -10 seconds, 67°C 15 seconds, 72°C 2.5 minutes, followed by 72°C for 10 minutes. PCR products were run on a 1% agarose gel stained with SYBR™ safe stain (Invitrogen) and the DNA bands at ~4600 bp were extracted using a Monarch® DNA . CC-BY-NC 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/798546 doi: bioRxiv preprint Gel Extraction Kit. Next, 1 μg of the PCR product was digested with KpnI (20 U) in NEB Buffer 1.1 at 37°C for two hours. Digested DNA was cleaned and concentrated with a Monarch® PCR and DNA Clean and Concentrate Kit and eluted into 20 μL nuclease free H2O. One microliter of the purified and digested DNA was ligated using T4 DNA ligase at ~25°C for 1 hour in standard conditions in a 20 μL reaction. Five microliters of the ligation reaction were transformed into chemically competent XL1-Blue E. coli via standard protocols. Cells were plated on LB agar containing 100 μg/mL carbenicillin and 50 μg/ mL kanamycin and grown at 37°C for ~16 hours. Cells were picked from transformation plates and grown in 50 mL TB with 100 μg/mL carbenicillin and 50 μg/mL kanamycin for ~12 hours, cells were pelleted, and DNA purified using compact midi-preps. . This oligo pool was used directly in NM without further amplification using 2 μg of the relevant golden gate plasmid as a template. Libraries were electroporated into high efficiency electrocompetent XL1-Blue E. coli (Agilent cat #: 200228) using 1 mm electroporation cuvettes at 1200V using a Eppendorf Eporator. Cells were plated on large bioassay plates (245mm x 245mm x 25mm, Sigma-Aldrich) containing LB agar + 100 μg/mL carbenicillin and 50 μg/ mL kanamycin and grown at 37°C for ~16 hours. Plates were scraped in 10 mL plain LB, broken into ~1.2 mL aliquots, pelleted, and stored -80°C. DNA was purified from 1x aliquot using a Monarch® Plasmid Mini-Prep Kit.